Method for the purification of 6-methoxy-1-phenazinol 5,10-dioxide

ABSTRACT

A novel improved procedure is described for the preparation of various metal complexes of 6-methoxy-1-phenazinol 5,10-dioxide by the reaction between 6-methoxy-1-phenazinol 5,10-dioxide and a metal salt or hydroxide in an aqueous suspension or a solution in aqueous mineral acid. Preferred metals disclosed as suitable for use in the process are calcium, strontium, magnesium and iron. Utility of the ferric chloride complex and the calcium complex in the purification of 6-methoxy-1-phenazinol 5,10-dioxide is also disclosed.

RELATED APPLICATION

This application is a division of application Ser. No. 213,748, filedDec. 29, 1971, and now abandoned, which was, in turn, acontinuation-in-part of application Ser. No. 139,287, filed Apr. 23,1971 and now abandoned.

BACKGROUND OF THE INVENTION

Metal complexes of 6-methoxy-1-phenazinol 5,10-dioxide (referred tohereinafter as myxin) heretofore were prepared by combining a saturatedsolution of myxin in a suitable organic solvent with a saturatedsolution of a metal salt in the same solvent. The precursor, myxin, isreadily prepared by selective alkylation of iodinin (1,6-phenazindiol5,10-dioxide).

Exemplary of the organic solvents generally employed in the priorprocesses used to prepare the metal complexes are acetic acid,acetonitrile, methanol, ether, chloroform, etc.

Preferably a solvent or solvent mixture in which both the myxin and themetal salts are more soluble than the metal complex formed by theirreaction is used in these processes since the complex is isolated byprecipitation from said solvents. This reaction may be carried out atroom temperature or, to facilitate solution of the reactants and reducethe amount of solvent needed, at temperatures above room temperature.

The metal complexes of myxin have a high degree and wide spectrum ofanti-microbial activity both in in vitro tests and in in vivo topicalinfections. In particular, the metal complexes have demonstrated a highlevel of activity against a wide variety of both gram positive and gramnegative bacteria, fungi, protozoa and helminths. This wide spectrum ofanti-microbial activity has manifested itself by the efficacy of themetal complexes as chemotherapeutic agents in combating topicalinfections.

There are, however, several drawbacks and deficiencies in the use oforganic materials as solvents in the preparation of the metal complexes.One evident drawback is, of course, the relatively large volume ofsolvent which must be used. In addition, the preferred solvent,acetonitrile, is flammable and its use in conjunction with the metalcomplexes is hazardous. Myxin itself is highly flammable and sensitiveto electrostatic discharge. Further, the metal complexes, on standing,have a tendency to form aggregates of from about 400 to about 600microns in size after isolation and purification from these organicsolvent systems. Finally, many metal complexes either cannot be preparedusing organic solvents in such procedures or, if prepared, cannot beisolated therefrom by crystallization.

In the use of myxin's anti-microbial activity, a high purity materialis, of course, advantageous. However, purification of myxin isexpensive, difficult, time consuming and requires elaborate equipment,i.e., column chromatography.

There is thus a need for a method which overcomes these drawbacks anddeficiencies. This inventin provides a method for the preparation andisolation of metal complexes of myxin which eliminates the problemsinherent in the use of organic solvents, and provides anon-agglomerating product in crystalline form.

In another aspect, this invention provides methods for utilizing boththe ferric chloride complex and the calcium complex of myxin to purifymyxin.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a novel method for the preparation of metalcomplexes of myxin using water or an aqueous mineral acid as thereaction medium. The invention also relates to the metal complexes perse, to compositions containing these metal complexes and to the use ofthese complexes both as chemotherapeutic agents in combating variousmicrobial infections and, in the case of the ferric chloride complex andthe calcium complex, as a means of purifying myxin.

DETAILED DESCRIPTION OF THE INVENTION

More specifically, this invention relates to a novel process for thepreparation of metal complexes of myxin by the reaction in water oraqueous mineral acids of myxin and the corresponding salt or hydroxideof a pharmaceutically acceptable metal. The phrase "pharmaceuticallyacceptable metal" as used herein denotes those metals which arenon-poisonous when utilized as part of a compound in a pharmaceuticalcomposition. Typical of such metals are zinc, iron, magnesium, calcium,aluminum, strontium and the like. The preferred metal complexes of thisinvention are those formed from calcium, magnesium, strontium and iron.

The novel process of this invention involves the reaction in water oraqueous mineal acids of myxin with a salt or hydroxide of the desiredmetal. The metal complexes can be isolated as crystals by precipitationfrom the reaction medium. The complex formed by the process of thisinvention can readily be prepared by simply combining an aqueoussuspension or a solution in aqueous mineral acid, at a temperature offrom about 20°C. to about 100°C., of myxin and a salt or a hydroxide ofa pharmaceutically acceptable metal. Where calcium and magnesium saltsor hydroxides are used in an aqueous suspension, the reaction takesplace in a solid state suspension since both reactants are eitherinsoluble in water or very slightly soluble therein. The insolublecomplex which forms is separated by filtration or precipitation.

Since myxin is very slightly soluble in water, myxin crystals are addedto water and suspended therein by agitation. Some myxin goes intosolution as evidenced by the light pink tinge which appears in thewater. While the concentration of myxin is not a critical factor per se,its limiting factor is the resultant increase in viscosity to anunsatisfactorily high level at a concentration of 100 grams per liter orhigher. The preferred concentration range is from 8-20 grams of myxinper liter of water.

Salts or hydroxides of the aforesaid metals can be used in this process.

The mole ratio of myxin to the metal salt or hydroxide should be aboutequimolar with, preferably, a slight excess of the metal salt orhydroxide, e.g., 0.75 moles of myxin per 1.25 moles of the metal salt,to insure reaction of all the myxin present in the aqueous suspension.

The reaction temperature and the reaction time are also not criticalfactors. The temperature can range from room temperature (20°-25°C.) upto 90°-95°C. and is selected based on other desiderata such as saltsolubility and reaction time. Completeness of reaction is noted by theappearance of a greenblack suspension (metal complex of myxin) and thecomplete absence of any red specks of myxin therein.

Myxin has a high degree and wide spectrum of anti-microbial activity inboth in vitro tests and in vivo topical infections. The metal complexesof myxin also exhibit this high degree and wide spectrum ofanti-microbial activity.

For example, the anti-microbial activity of calcium myxin dihydrate,prepared by the method of this invention, is evaluated and compared tothat of myxin itself both in vivo against 4 strains of bacteria and invitro against yeast and fungus infections. The table below shows thisanti-microbial efficacy.

    ______________________________________                                        Anti-microbial Activity of Myxin and Calcium Myxin Dihydrate                  Prepared in Water                                                                        In Vivo                                                            Organism   ED.sub.50.sub.' mcg/ml                                                      Calcium Myxin                                                                 Dihydrate   Myxin                                                    ______________________________________                                        S. agalactiae                                                                            <4             1                                                   S. aureus  <20           13                                                   E. coli    60             8                                                   P. aeruginosa                                                                            20            30                                                            In Vitro                                                                      Minimum Inhibitory Concentration, mcg/ml.                                       Calcium Myxin                                                      Organism   Dihydrate     Myxin                                                ______________________________________                                                   static   cidal    static cidal                                     C. albicans                                                                              <0.8     <0.8     1.8    23                                        M. canis   <0.8     <0.8     0.9    0.9                                       ______________________________________                                    

When compounds of this invention are employed in the treatment ofmicrobial infection they are conveniently utilized in compositions withsuitable carrier materials for use as chemotherapeutic agents incombating mammalian disease. They are formulated by uniformlydistributing in a vehicle that is chemically compatible with theparticular compound, non-inhibiting with respect to the activeingredient and essentially non-injurious to body tissue under theconditions of use. When formulated into compositions suitable fortopical administration, the novel compounds of this invention arepreferably employed in amounts ranging from about 0.1% to about 1.5% byweight of the chemotherapeutic composition, e.g., gel, cream, ointment,suspension, suppository or the like. It will be understood that thecompounds of this invention, when employed in forms suitable for topicaladministration may be utilized in diverse formulations: for example,solid formulations including finely divided powders and granularmaterials, liquid formulations including suspensions, concentrations,slurries, tinctures, aerosols and the like, depending on the applicationintended and the formulation media desired. They may be employed ascreams, gels, jellies, ointments, pastes, etc. Formulations containingingredients which have reactivity with the complexes of this inventionmay alter the mole ratios of the components of the complex since suchingredients can be incorporated into the complex as ligands. Thisinvention is intended to include such compositions.

In the preparation of myxin, the crude product is contaminated with, forexample, solvent, unreacted iodinin starting material and suchby-products and degradation products as 6-methoxy-1-phenazinol-10-oxide;1,6-dimethoxyphenazine 10-oxide and 1,6-dimethoxyphenazine 5,10-dioxide.Heretofore purification of this crude product was achieved by suchtedious and timeconsuming procedures as column chromatography.

Using the ferric chloride complex prepared by the process of thisinvention, however, it has been found that myxin purification can beachieved in a simple process involving the preparation and isolation ofthe ferric chloride complex from crude myxin followed by the breaking ofthe complex with acetone to yield a pure myxin. This myxin purificationis specific to the ferric complex made by the process of this inventionsince processes for complex formation using organic solvents require apure myxin as the starting material.

In addition, in the purification of myxin by the above ferric complexmethod, a crude myxin residue wherein the main impurity is1,6-dimethoxyphenazine 5,10-dioxide (dimethoxy iodinin) remains.Formation, by the process of this invention, of the calcium complex ofmyxin from this crude myxin residue wherein the dimethoxyiodinin remainsunchanged, isolation of the complex from the dimethoxyiodinin andsubsequent breaking of the calcium complex yields pure myxin.

The following examples illustrate the invention.

EXAMPLE 1 Preparation of 6-methoxy-1-phenazinol 5,10-dioxide, calciumdihydrate complex

To 10 liters of distilled water heated to 50°C. are added, withstirring, 16 grams of calcium hydroxide. After 10 minutes, 51.5 grams ofmyxin are added with vigorous stirring. The reaction mixture is heatedto 80°C. and maintained at that temperature under agitation for 2 hours.The mixture is then cooled to room temperature. The black solid whichprecipitates is filtered, washed with distilled water (2 × 1,000 ml.)and acetone (3 × 1,000 ml.) dried at 55°-60°C. and 0.1 mm for 20 hours.The resulting calcium myxin dihydrate is a crystalline powder having thefollowing characteristics:

Calculated for C₂₆ H₂₂ N₄ O₁₀ Ca: C, 52.88; H, 3.75; Ca, 6.77

Found: C, 53.07; H, 3.58; Ca, 6.83

Visible ultraviolet absorption maxima (in 0.1N methanolic HCl):

    512 nm a        25.2                                                          355 nm a        16.7                                                          286 nm a        308.7                                                     

EXAMPLE 2 Preparation of 6-methoxy-1-phenazinol 5,10-dioxide, strontiumdihydrate complex

To 500 milliliters of distilled water are added, with stirring, 9 gramsof strontium hydroxide octahydrate. After 10 minutes, 9 grams of myxinare added with vigorous stirring. The reaction mixture is heated to80°C. and maintained at that temperature under agitation for 2 hours.The mixture is then cooled to room temperature and the finely dividedsolid, i.e., strontium myxin, is filtered, washed with distilled water(3 × 500 ml.), acetone (2 × 500 ml.), methylene chloride (2 × 500 ml.)and acetone (1 × 500 ml.). The product is dried at 60°C. and 0.1 mm for20 hours. The resulting strontium myxin dihydrate is characterized asfollows:

Calculated for: C₂₆ H₂₂ N₄ O₁₀ Sr: Sr, 13.73;

Found: Sr, 14.22

EXAMPLE 3 Preparation of 6-methoxy-1-phenazinol 5,10-dioxide, ferricchloride complex

To 250 ml. of aqueous hydrochloric acid (37%), 10 grams of myxin areadded, with stirring. After 10 minutes, 10 grams of ferric chloridehexahydrate are added with agitation continued for 16 hours. Theresulting black solid is filtered, washed with hydrochloric acid (1 ×100 ml.), acetic acid (1 × 100 ml.), and methylene chloride (2 × 500ml.). The product is then dried at 60°C. and 0.1 mm for 20 hours. Theresulting ferric chloride myxin complex is characterized as follows:

Calculated for: C₁₃ H₁₀ N₂ O₄.HCl.FeCl₃ : Fe, 12.22; C, 34.17; H, 2.43;N, 6.13

Found: Fe, 12.22; C, 34.39; H, 2.52; N, 6.26.

EXAMPLE 4 Preparation of 6-methoxy-1-phenazinol 5,10-dioxide, magnesiumdihydrate complex

To 10 liters of distilled water preheated to 50°C., 12.8 grams ofmagnesium hydroxide is added with stirring. After 10 minutes, 51.5 gramsof myxin is added with vigorous stirring. The reaction mixture is thenheated to 80°C. and maintained at that temperature under agitation for 2hours. The mixture is then cooled to room temperature. The resultingblack solid precipitate is filtered, washed with distilled water (2 ×1,000 ml.) and acetone (3 × 1,000 ml.) and dried at 55°-60°C. and 0.1 mmfor 20 hours. The product, magnesium myxin dihydrate, is characterizedas follows:

Melting point 142° (with decomposition)

Calculated for: C₂₆ H₂₂ N₄ O₁₀ Mg: C, 65.36; H, 3.83; Mg, 4.18

Found: Mg, 4.22.

EXAMPLE 5

5.6 kg. of a 30% solids paste, from a preparatory procedure for myxinand containing 60% myxin, are charged to a reaction kettle containing 29liters of concentrated hydrochloric acid, stirred for 30 minutes duringwhich a blue color believed to result from a protonated myxin complex inthe acid develops and then filtered to remove undissolved impuritiesusing a two-inch layer of Hyflo (an inert silica). The filter cake iswashed with 3 liters of concentrated hydrochloric acid to remove anyuncomplexed myxin thereon and the acid solution is returned to thereaction kettle. One kilogram of ferric chloride hexahydrate is added tothe kettle with stirring for one hour. The black solid ferric chloridecomplex of myxin which forms is vacuum filtered and washed with 4 litersof acetic acid and then with 8 liters of chloroform.

The ferric chloride complex is returned to a reaction kettle containing20 liters of acetone at 10°C. and stirred overnight at that temperature.The resulting crystallized myxin is separated by filtration, washeduntil neutral with water followed by an acetone wash, and dried undervacuum at 60°C.

The acetone filtrates are combined, diluted with water, extracted withmethylene chloride and concentrated under vacuum to yield additionalmyxin as a crude product containing approximately 70% of myxin and suchimpurities as 6-methoxy-1-phenazinol and, mainly, dimethoxyiodinin,1,6-dimethoxyphenazine 5,10-dioxide.

EXAMPLE 6

2.0 kg. of crude dry myxin, from a preparatory procedure for myxin andcontaining 65% myxin, is added with stirring to a reaction kettlecontaining 60 liters of 50% aqueous sulfuric acid at 25°C. Stirring iscontinued for 30 minutes and then the solution is filtered through a2-inch layer of Hyflo to remove undissolved impurities. The filter cakeis washed with 4 liters of 50% sulfuric acid to remove any uncomplexedmyxin thereon while retaining unreacted iodinin.

The blue-colored filtrate is returned to the reaction kettle. Two kg. offerric chloride hexahydrate is added to the kettle with stirring for 1hour. The resulting black solid is vacuum filtered and washed with 8liters of acetic acid followed by 16 liters of chloroform.

The black ferric complex is returned to the kettle containing 30 litersof acetone and stirred for 20 hours at 10°C. The resulting crystallizedmyxin is separated by filtration, washed with water and with acetone anddried under vacuum at 60°C.

The acetone solutions are combined, diluted with water, extracted withmethylene chloride, and concentrated under vacuum to yield additionalmyxin as a crude product wherein the main impurity is dimethoxyiodinin.

EXAMPLE 7

This example illustrates the use of the calcium complex of myxin,prepared by the process of this invention, in the purification of thecrude myxin product, rich in dimethoxyiodinin, obtained from thepurification processes described in Examples 5 and 6 above.

To a 12-liter flask containing 10 liters of water and 200 g. of calciumhydroxide are added 200 g. of the second crude myxin product, containing20-25% of dimethoxyiodinin, obtained as the residue of the process ofExample 6. The mixture is stirred for 2 hours at 80°C., cooled to roomtemperature and vacuum filtered. The solid is washed with cold(20°-25°C.) acetone and then stirred with two liters of boiling acetone.This washing procedure is repeated until, as determined by Triple LayerChromatography, all of the soluble orange-colored dimethoxyiodinin iswashed from the solid black calcium complex of myxin.

The calcium complex is added to 6 liters of 10% hydrochloric acid andstirred for 1 hour. Pure myxin, which separates as a red solid, isseparated by filtration, washed with water until neutral and then withacetone, and dried under vacuum.

I claim:
 1. A process for preparing pure 6-methoxy-1-phenazinol5,10-dioxide which comprisesa. reacting a ferric chloride hexahydrate ina suspension of a crude mixture containing 6-methoxy-1-phenazinol5,10-dioxide in a concentrated aqueous mineral acid; b. isolating thesolid ferric chloride complex of 6-methoxy-1-phenazinol 5,10-dioxidewhich forms; C admixing this complex with acetone, thereby breaking thecomplex; c. d. recovering the pure 6-methoxy-1-phenazinol 5,10-dioxide,wherein the concentrated aqueous mineral acid is selected from the groupconsisting of hydrochloric acid and sulfuric acid.
 2. A process asdescribed in claim 1 wherein the acid is hydrochloric acid.
 3. A processas described in claim 1 wherein the acid is sulfuric acid.